T-jump study of calcium binding kinetics of calcium chelators.
Recent experimental studies have investigated the kinetic competition between calcium chelators and the secretion apparatus at a fast central synapse. Simultaneously, mathematical modelling studies indicate the importance of a quantitative knowledge of the binding kinetics of the chelators in studying fast physiological processes operating on a millisecond time scale. Using the temperature-jump relaxation method, I have studied the in vitro kinetics of Bis-Fura-2, Furaptra, Fluo-3, Calcium-Green-1, Calcium-Green-5N, Calcium-Orange-5N as well as EGTA, BAPTA and H-EDTA in conditions which are identical to those implemented in our patch clamp recordings, i.e. 100-140 mM CsCl, 20-40 mM Cs-HEPES, 8 mM NaCl, pH = 7.2 at 22 degrees C. The results can be summarized as follows: all fluorescent indicators have on rates in the range of 10(8)-10(9) M-1s-1. They differ significantly with respect to their off-rates from each other according to their affinities, ranging from 100 s-1 up to 26,000 s-1. BAPTA is kinetically almost indistinguishable from Fura-2. EGTA and H-EDTA have small binding rate constants for calcium in the range of 3 x 10(6) M-1s-1 since, at pH 7.20, protons need to be dissociated from the chelators before they can bind calcium ions.
Naraghi
M
M
Department of Membrane Biophysics, Max-Planck-Institute for Biophysical Chemistry, Göttingen, Germany. mnaragh@gwgd.de
D000814:Aniline Compounds / Q000378:metabolism; D002118:Calcium / Q000378:metabolism*; D002614:Chelating Agents / Q000378:metabolism*; D004492:Edetic Acid / Q000378:metabolism; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000378:metabolism; D005456:Fluorescent Dyes / Q000378:metabolism*; D016257:Fura-2 / Q000031:analogs & derivatives / Q000378:metabolism; D007202:Indicators and Reagents / Q000378:metabolism; D009930:Organic Chemicals; D004369:Pentetic Acid / Q000378:metabolism; D013696:Temperature; D014966:Xanthenes / Q000378:metabolism
D000814:Aniline Compounds; D002614:Chelating Agents; D005456:Fluorescent Dyes; D007202:Indicators and Reagents; D009930:Organic Chemicals; D014966:Xanthenes; C058776:2-(2-(5-carboxy)oxazole)-5-hydroxy-6-aminobenzofuran-N,N,O-triacetic acid; C071336:calcium green; C071335:calcium orange; C059715:Fluo-3; D004533:Egtazic Acid; D004369:Pentetic Acid; D004492:Edetic Acid; C025603:1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; D002118:Calcium; D016257:Fura-2